The emergence of novel GII.4 norovirus variant, Sydney_2012, in Shanghai, China / 病毒学报

To describe the epidemiological characteristics of norovirus (NOV) associated acute gastroenteritis in Shanghai and characterize the evolution pattern of circulating strains. From March 2012 to February 2013, 502 stool specimens were collected from adult (> or = 16 years) outpatients who visited either of the two sentinel hospitals in Shanghai for acute gastroenteritis. Molecular detection and genotyping of NoV were performed and the phylogenetic relationship of the circulating strains has also been comprehensively analyzed. The epidemics level of GI NoV was low throughout the surveillance period, with the positive rate of 3.78% (19 cases), and no seasonality of GI NoV infection could be distinguished. For GII genogroup, higher epidemics in adults in Shanghai, with the detection rate of 17.13% (86 cases), were observed. And relatively high epidemics of GII NoV infection were spotted between October and December in 2012. The frequency of NoV associated acute gastroenteritis in older people is significantly higher than that in young individuals (P < 0.05). Sequencing and genotyping analysis revealed that the high epidemics of GII NoV infection between October and December in 2012 is associated with the emergence of a novel GII.4 norovirus strain, termed Sydney_2012. Sequence analysis also demonstrated that this was a recombinant virus between a GII.e polymerase and GII.4 capsid, which has also been the dominant circulating strain in Shanghai. In 2012, a new GII.4 variant, termed Sydney_2012, emerged in Shanghai and caused high epidemics of acute gastroenteritis during late autumn and winter.

Plasma proteome analysis of chronic hepatitis B patients with different stages of liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify plasma biomarkers with specific relation to the various liver fibrosis stages that can be used to assess hepatitis B virus (HBV)-infected patients for non-invasive liver fibrosis and to evaluate the prognosis of the liver fibrosis.</p><p><b>METHODS</b>Plasma samples were collected from 80 HBV-positive patients at the Hepatitis Department of Shanghai Public Health Clinical Center between September 2008 and January 2011. The samples were grouped according to the patient's stage of hepatic fibrosis determined by liver biopsy: S0-1 (n = 40), S2-3 (n = 20), and S4 (n = 20). Each plasma sample was processed to remove the two most abundant proteins, albumin and immunoglobulin G (IgG), and then resolved by two-dimensional (2D) electrophoresis. ImageMaster 2D analysis software was used to identify differentially expressed proteins related to the liver fibrosis stages. After trypsin digestion, the differential proteins were identified by online reversed-phase nano-flow liquid chromatography coupled with electrospray ionization ion trap mass spectrometry (MS).</p><p><b>RESULTS</b>The patients in the three groups were not significantly different in age (range: 30-50 years; P = 0.053) or sex (x² = 0.155, P = 0.926). Low-abundance proteins were efficiently enriched by the albumin/IgG depletion method. Fourteen differentially expressed proteins were detected among the S0-1, S2-3 and S4 groups, all of which were identified by tandem MS and included fibrinogen gamma chain, haptoglobin, complement C3, Ig kappa chain C region, and apolipoprotein A-I.</p><p><b>CONCLUSION</b>Plasma proteomic analysis of chronic hepatitis B patients identified a panel of differentially expressed proteins related to different stages of liver fibrosis. These proteins may represent diagnostic and prognostic biomarkers of HBV-related hepatic fibrosis.</p>

Subcellular proteome analysis of immune or alcohol induced rat liver fibrosis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the mechanism of liver fibrogenesis and to find new non-invasive biomarkers.</p><p><b>METHOD</b>In this study, we used subcellular proteomic technology to study the plasma membrane proteins related to immune or alcohol induced liver fibrosis. Rat liver fibrosis models were induced by pig serum or alcohol injection. The liver fibrogenesis were detected by James's staining in the rat models after 2, 4, 6 and 8 weeks of treatment. The liver plasma membrane (PM) of the 2- and 8-week treatment model rats were enriched by two-step sucrose density gradient centrifugation. The purity of PM was verified by western blotting, and the plasma membrane proteins were extracted and analyzed by 2 DE. The differentially expressed proteins were identified by LC-MS/MS. Cellular location and function of these identified differential protein were classified.</p><p><b>RESULTS</b>Immune or alcohol induced liver fibrosis rat models were successfully established. Liver plasma membrane was significantly enriched after sucrose density ultracentrifugation treatment. 87 differential protein spots were find out by 2DE combined with LC-MS/MS from the liver plasma membrane proteins of the 2- and 8-week treatment rat models, which corresponded to 30 non-redundant proteins including annexin A2, keratin 8 and keratin 18.</p><p><b>CONCLUSIONS</b>A list of differentially expressed proteins relate to liver fibrosis were successfully identified. Differential proteins such as annexin A2, keratin 8 and keratin 18 could be new biomarkers for liver fibrosis diagnosis.</p>

Immune response for phase I clinical trial of a hepatitis B immunogenic complex therapeutic vaccine, YIC / 中华肝脏病杂志

<p><b>OBJECTIVE</b>A hepatitis B immunogenic complex therapeutic vaccine, yeast-derived recombinant HBsAg combined with human anti-HBs immunoglobulin (YIC), was evaluated for safety and immune response in phase I clinical trial.</p><p><b>METHODS</b>The subtypes IgG1, IgG2, IgG3 and IgG4 of serum anti-HBs collected from 20 immunized subjects were analyzed by ELISA. The lymphocyte proliferation assay was carried out in five subjects and was analyzed by 3H-thymidine incorporation. The assays for IFNgamma, IL-2, IL-4, IL-6, IL-10 and TNFalpha were measured using Human Cytometric Bead Array Kit with FACSCalibur.</p><p><b>RESULTS</b>The results showed that the subtypes of anti-HBs antibodies induced by 30, 60 and 90 microg YIC-immunized groups among all of the adult volunteers (20/20) were IgG1 and IgG3. The level of IgG1 was higher than that of IgG3 in each volunteer but the strength was different from each other. The rHBsAg-stimulated lymphocyte proliferation induced by three injections of 90 microg of YIC showed that the stimulation index was more than 2.0 in four out of the five individuals (4/5), ranging from 2.70 to 4.75. PHA-stimulated lymphocyte proliferation was not related to rHBsAg-stimulated lymphocyte proliferation. In the 60 microg YIC-immunized group there was no significant difference between the levels of IFNgamma, IL-2, IL-4, IL-6 and IL-10 at day 0 and day 42. At day 71, in comparison to day 0, the level of IFNgamma was higher in all eight subjects studied (P = 0.015) and the level of IL-2 was also increased in seven out of eight subjects (P = 0.002). In contrast, the levels of IL-4, IL-6, IL-10 and TNFalpha showed no significant difference in all the subjects (P-values: 0.298, 0.976, 0.202 and 0.996).</p><p><b>CONCLUSION</b>Our results indicate that this hepatitis B immunogenic complex therapeutic vaccine (YIC) can induce a potent anti-HBs response.</p>

Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To construct the full-length complementary DNA of HCV genome from an HCV infected patient.</p><p><b>METHODS</b>Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system.</p><p><b>RESULTS</b>The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media.</p><p><b>CONCLUSION</b>These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.</p>

Experimental study of K562 cell apoptosis induced by siRNA / 中华血液学杂志

<p><b>OBJECTIVES</b>To construct a siRNA expression vector pBCR6 that produces siRNA against bcr/abl mRNA and detect apoptosis rate of K562 cells after pBCR6 transfection.</p><p><b>METHODS</b>Template sequence for siRNA was designed, synthesized and inserted into an expression vector pSilencer1.0-U6. Restriction analysis and sequencing were performed to verify the pBCR6 vector. Then pBCR6 was transfected into K562 cells by X-tremeGene Q2. pSilencer1.0-U6 was used as the control. At different time point after transfection, apoptosis rate was determined by Tunel and Annexin V+ PI with FCM.</p><p><b>RESULT</b>pBCR6 was verified by restriction analysis and sequencing. The apoptosis rate of K562 cells markedly increased at 48 and 72 hour after transfected with pBCR6, and increased in a time-dependent manner [the apoptosis rate of transfected K562 cells was (47.80 +/- 1.63)% at 72 hrs, whereas the control group was (6.67 +/- 0.37)%, P < 0.0001] No prominent change in apoptosis rate was found in the control.</p><p><b>CONCLUSION</b>The siRNA expression vector against bcr/abl mRNA was successfully constructed. The pilot study showed that pBCR6 could effectively induce K562 cells apoptosis. siRNA may be a new tool for molecular target therapy for chronic myelogenous leukemia.</p>